DHEA is considered an ‘antidote for aging’ and exerts protective effects against a variety of diseases associated with aging, such as atherosclerosis (AS) [21], osteoporosis (OP) [22] and dementia [23]. OA is also considered an aging-related disorder; however, data concerning the mechanism of DHEA in OA are still limited. Our previous histomorphological study demonstrated the beneficial structure-modifying properties of DHEA on rabbit cartilage in vivo [12]; however, the exact molecular mechanism behind this chondroprotection is still unclear. Recent evidence showed the crucial role of ERK1/2 signaling in the onset of OA and ADAMTS-mediated degradation [14] and the potential of DHEA to inhibit human osteoclastic resorption in an ERK1/2 signaling-dependent manner [19], thus slowing the progress of OP; therefore, we hypothesized that DHEA might slow osteoarthritic cartilage degradation via ERK1/2 signaling in our latest review article [24]. In the current study, we aimed to explore this hypothesis in osteoarthritic chondrocytes.
Aggrecan is the major proteoglycan in cartilage. Aggrecan degradation, which has been attributed to the action of aggrecanases, is an important manifestation of OA [25]. These enzymes have been identified as the proteinases responsible for cleaving the matrix proteoglycan aggrecan at multiple positions; this activity is considered a hallmark of cartilage degradation during inflammatory joint diseases such as OA [26]. Several members of the ADAMTS family of proteins have been shown to cleave aggrecan in vitro at the aggrecanase cleavage site [27–29]. Of these, ADAMTS-4 and ADAMTS-5 are the most efficient aggrecanases and have generally been considered the most likely candidates for a role in the pathogenic mechanisms of OA [30–31]. Therefore, inhibition of ADAMTS-4/5 might be an effective therapeutic strategy for OA. Indeed, increasing evidence has shown that the deletion of ADAMTS-4/5 provided significant protection against proteoglycan degradation ex vivo and decreased the severity of OA [32–35]. In the current study, we found that DHEA suppressed the mRNA expression of ADAMTS-4 and ADAMTS-5 and enhanced the mRNA expression of aggrecan and collagen type 2 in OA chondrocytes. Additionally, we used an ARGxx antibody, which does not react with intact aggrecan, to detect the aggrecanase-generated aggrecan fragments. The 374ARGS epitope is generated by aggrecanase cleavage at TEGE373-374ARGS [36]; thus, the assay is specific for aggrecanase-mediated aggrecan degradation [37]. Our results showed a reduction in the protein level of ARGxx in the DHEA group, indicating reduced activity of ADAMTS-4/5 in the DHEA group. This finding was consistent with our RT-PCR data.
Many signaling pathways are known to be involved in cartilage degradation in OA; of these, we focused on the ERK1/2 signaling pathway because of its importance in OA progression [38–39]. For example, the ERK1/2 signaling pathway has been reported to induce ADAMTS-4 and ADAMTS-5 expression, leading to ECM degradation [40]. In the present study, we also demonstrated that IL-1β stimulation induced the phosphorylation of ERK1/2 and enhanced the expression of downstream catabolic genes, including ADAMTS-4 and − 5, while DHEA reduced ERK1/2 phosphorylation and rescued ADAMTS-derived aggrecan degradation and proteoglycan loss in chondrocytes. To further explore the role of ERK1/2 signaling in DHEA-mediated protection against OA, we blocked ERK1/2 signaling with the specific inhibitor PD98059 in DHEA-treated chondrocytes. Blocking ERK1/2 signaling significantly enhanced the protective effects of DHEA, as indicated by reduced ADAMTS-4 and − 5 expression and increased aggrecan and collagen type 2 expression. These results suggest that the chondroprotective effect of DHEA is partly mediated through the ERK1/2 signaling pathway.
There are some limitations to this study. Apart from biochemical factors such as proinflammatory cytokines (IL-1β and TNF-α) [41], biomechanical factors are other crucial contributors to the onset of OA [42]. It is well known that this chronic joint disease is characterized by impairments in the loadbearing function of articular cartilage [43]. In the current study, we applied IL-1β as a biochemical stimulus to establish the osteoarthritic phenotype in chondrocytes and explored the mechanism of DHEA against OA; however, we do not know whether this chondroprotective effect of DHEA occurs under abnormal biomechanical stimuli and whether the responsible upstream signaling pathway is the same or involves other pathways. To address this limitation, future research should focus on chondrocyte metabolism induced by abnormal biomechanics and the effect of DHEA on these chondrocytes. Encouragingly, there are new methods that can achieve these goals. For instance, the Flexcell tension system could exert cyclic mechanical stretch on cultured chondrocytes [44–45]. Collagen-coated polyacrylamide gels have been used as substrates to culture chondrocytes. The substrate stiffness could be varied by changing the bisacrylamide concentration, thus loading different stresses on chondrocytes [43, 46].
In conclusion, our results demonstrated that DHEA inhibited IL-1β-induced ADAMTS-4 and ADAMTS-5 expression by suppressing the ERK1/2 signaling pathway in rabbit primary chondrocytes. Overall, these results suggest that DHEA may be a potent anti-osteoarthritic agent for the treatment of OA.